ARTICLE   Open Access    

Exogenous hydrogen sulfide enhanced Al stress tolerance in tea plant Camellia sinensis

  • # Authors contributed equally: Anqi Xing, Zaifa Shu

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  • Al is an essential element for the growth of tea plants roots, but excessive Al affects growth and development of Camellia sinensis. The underlying mechanism, particularly regulation of gas signaling molecule H2S, remains unclear. This study aims to uncover the function of H2S on C. sinensis under Al stress by treating hydroponic tea seedlings with different Al concentration, Na2S (H2S donor) and DL-propargylglycine (PAG, synthesis inhibitor). High concentration of Al inhibits growth of tea roots, while H2S significantly improves the effects caused by Al stress. Whether it is 2 mM Al3+ or 4 mM Al3+, H2S reduces content of Al in the entire plant and roots, increases root activity, further promotes root growth, increases fresh and dry weight, regulates ion homeostasis, improves cell structure, increases chlorophyll content, and thus reduces the damage of Al toxicity in C. sinensis. Moreover, in response to the stress of 2 mM Al3+, H2S simultaneously alleviates Al stress by regulating substances related to antioxidant pathways, increasing content of GSH and GSSG, enhancing activity of GST, GR, LCD, and key components of tea, in order to alleviate Al stress. These approaches have effectively improved Al tolerance of C. sinensis, providing a new perspective for the study of H2S enhancing Al tolerance.
  • The Trichoderma genus encompasses a wide-ranging collection of filamentous fungi, prevalent in various natural ecosystems[1]. Trichoderma species within this genus have earned acclaim for their exceptional capacity to inhabit plant roots, stimulate plant growth, and showcasing biocontrol attributes against a spectrum of fungal adversaries[2]. Employing tactics like mycoparasitism, antibiosis, competitive resource acquisition, and plant resistance induction, these species effectively manage fungal diseases[3]. Notably, they are increasingly utilized in agriculture as biofertilizers and biopesticides[1]. Trichoderma-based bio-fungicides, available in different formulations like wettable powders, granules, and flowable concentrates, offer a convenient application to seeds, seedlings, soil, and foliage[4,5]. Besides their disease-fighting properties, these bio-fungicides promote plant growth through various mechanisms such as phytohormone production, nutrient solubilization, and stress tolerance enhancement[3]. Recent progress in Trichoderma-based formulations has led to innovative materials, advanced nanotechnology strategies, and genetic engineering techniques aimed at boosting stability, shelf life, and efficacy[4]. Among these advancements, biochar has shown promise as an ideal carrier for Trichoderma formulations due to its high porosity, surface area, and soil stability maintenance abilities[6]. New research indicates that biochar can strengthen Trichoderma's biocontrol properties[7,8]. Experiments show that using Trichoderma bio-fungicides on soil blended with biochar is more effective in fungal disease control than on unamended earth[9]. Likewise, applying these bio-fungicides on biochar-coated seeds provides better resistance against fungal diseases in seedlings[7,10]. Such biotechnological advancements in Trichoderma-based formulations can promote sustainable agricultural practices by reducing reliance on chemical pesticides[11]. This, in turn, helps mitigate the ecological impact of agricultural activities and enhances food and feed safety[12]. By protecting plants from fungal diseases and improving soil fertility, Trichoderma-based bio-fungicides hold promise for enhancing crop yield[1]. Trichoderma formulations play a crucial role in minimizing harm to non-target organisms while maximizing the effectiveness of the active ingredient[13]. While Trichoderma is significant in ensuring agronomic safety, challenges in their formulation persist due to potential degradation of the biomass or bioactive metabolite caused by factors like exposure to air, light, and temperature[14]. Additionally, these products need to be easy to handle, apply, and produce[15,16]. To address this objective, the present study aims to offer a comprehensive examination of various technological advancements that enhance the efficiency of natural preparations. Distinguishing itself from typical literature reviews that predominantly delve into the biological attributes of metabolites, this review incorporates a bibliometric analysis of biopesticides and their formulations[17]. This analysis employs quantitative and statistical indicators to identify patterns related to the most critical pest issues, agriculture's susceptibility, sources of biological control, innovative methodologies, and the current status of Trichoderma formulations. The insights presented in this analysis significantly contribute to the bibliometric methodology, potentially promoting positive strides in the advancement of technology for Trichoderma formulation. Additionally, it offers valuable suggestions for researchers engaged in this field.

    Bibliometric analysis is a technique employed to scrutinize the characteristics and evolving patterns within academic literature using various mathematical and statistical methods[10]. Through this approach, we can quantitatively assess the overall state of the literature, collaborative relationships, research areas of interest, and the development trends in a specific research field[18]. A descriptive analysis of the corpus of published research pertaining to Trichoderma formulations was conducted. This analysis entailed the examination of co-occurring terms within the body of published articles, allowing for the elucidation of evolutionary trends in scientific themes[19]. The fundamental aim of this research is to conduct an exhaustive review of the existing body of literature on Trichoderma formulations and to project the areas of highest interest and potential for future investigation[20]. One of the primary objectives of bibliometric analysis is to assess the trends in research related to Trichoderma formulations, and to identify the most influential authors and institutions in the field of Trichoderma research. Determining the impact of research in terms of citations, patents, or applications in real-world scenarios.

    As a result, this study seeks to investigate the following research objectives: In the field of Trichoderma formulations, what are the key research themes and trends observed from 2016 to 2023 include:

    (1) How is research on Trichoderma formulations distributed geographically, and what regions exhibit the most active contributions to the field? (2) Can bibliometric analysis predict future trends and potential innovations in Trichoderma formulations research based on historical patterns? (3) The article follows a well organised structure[21]. Initially the research methodology adopted for the study is outlined. Subsequently, a well-organized article is crucial for effectively communicating research findings to the intended audience[22].

    Literature retrieval was performed online through the Science Citation Index Expanded (SCI-E) of the Web of Science Core Collection (WoSCC, Clarivate Analytics) from 2016 to 2023[21,22]. Scopus is a preferred data source for bibliometric analysis, and it provides comprehensive information and data from a multi-disciplinary field of literature[23]. To retrieve literature comprehensively and accurately on Trichoderma formulations, different search terms and retrieval strategies were assembled in this study. Finally, the optimal search items were set as follows: TS = ('Trichoderma formulation*') OR ('Bio formulation of Trichoderma*') OR ('Bio control') OR ('Antagonist') OR ('Rhizosphere fungus')[24]. The data range was set from 2016 to 2023, to collect all relevant publications. It is worth noting that as the Scopus database data network is constantly updated, the results may vary depending on the exact retrieval date.

    A detailed literature retrieval process was performed online through Science Citation Index Expanded (SCI-E) of the Web of Science Core Collection (WoSCC, Clarivate Analytics) from 2016 to 2023, and considered Scopus as a preferred data source for bibliometric analysis. Additionally, we outlined the search terms and retrieval strategies that are used in our study and set the range of data from 2016 to 2023 to collect all relevant publications. If we sum up our descriptions narrate on the following key points, like data sources, search terms, data range, constantly updated data base, and optimal search items[25]. The search strategy was designed to capture relevant literature on Trichoderma formulations. The search terms included Trichoderma formulation, bio-formulation of Trichoderma, biocontrol, antagonist and Rhizosphere fungus. The asterisks, in the search terms are used as wildcard characters to capture different word endings. The data range was set from 2016 to 2023 to collect all relevant publications within that timeframe. This was the period during which the literature retrieval was performed[25]. It is mentioned that as the Scopus database data network is constantly updated, and the results may vary depending on the exact retrieval date. This indicates that the study acknowledged the dynamic nature of the database and its potential impact on the results[26]. After assembling different search terms and retrieval strategies, the study determined the optimal search items, which were the selected search terms that would yield the most comprehensive and accurate results for the study's objectives. Overall, the present study took a systematic approach to literature retrieval, considering multiple data sources and employing a combination of search terms to ensure the retrieval of relevant publications on Trichoderma formulations. It is worth noting that as the Scopus database data network is constantly updated, to add upon, the results may vary depending on the exact retrieval date.

    To ensure the credibility of the research conclusions, this study gathered peer-reviewed English journal articles to summarize global research perspectives. It's important to mention that articles not aligned with this paper's purpose were manually omitted in the final phase of data collection[27]. For instance, some articles explored the relationship between plants and microorganisms on leaves. Eventually, a total of 287 articles that met all criteria were sourced from Scopus. These pieces represented almost all top-tier experimental studies on Trichoderma formulations from 2016 to 2023 worldwide. The variability among these articles could effectively indicate the trend of related research development. Therefore, these publications were prioritized for further analysis and assessment. Figure 1 illustrates the flowchart of the literature retrieved in this study[28].

    Figure 1.  Flow chart of literature review methodology.

    Table 1 presents the top 10 countries/regions, institutions, authors, and journals that published the most studies on Trichoderma formulations. As indicated in Table 1, China emerged as the country making the most significant contribution, with 1,231 publications, accounting for 20.33% of the total. India and Pakistan followed closely, ranking second and third, with 1,096 (18.10%) and 618 (10.20%) publications, respectively. Among the institutions, the Chinese Academy of Sciences held the top spot, boasting 160 (2.64%) publications. Following closely was the University of Agriculture, Faisalabad (144, 2.37%), and Nanjing Agricultural University (137, 2.26%). In the realm of scholarly contributions, prolific authors often set the tone for research trends. Identifying these influential scholars can shed light on the direction of the research field[29]. The leading author in the study of rhizosphere microorganisms was Wang Y, with 102 publications. Li Y, Zhang Y, and Hkan M were also highly prolific, each publishing nearly 90 studies. These works were predominantly featured in prominent journals in Ecology and Botany, such as Frontiers in Microbiology (3.73%), Frontiers in Plant Science (2.36%), and Plant and Soil (2.03%).

    Table 1.  Leading journals contributing to the existing body of knowledge in the field of formulation of Trichoderma.
    Sl. no. Name of journal No. of publication Citations
    1 Journal of Applied Microbiology 2 40
    2 Applied Microbiology and Biotechnology 2 16
    3 Indian Phytopathology 3 2
    4 Biological Control 2 59
    5 Crop Protection 2 30
    6 Frontiers in Microbiology 2 23
    7 Journal of Biological Control 2 1
    8 Medicinal Plants 2 5
     | Show Table
    DownLoad: CSV

    The analysis of scientific production on Trichoderma formulations demonstrated the trend of publications per year on Trichoderma formulation studies. It was observed that published research showed a significant increase of 71.24% over the last decade (2016–2023) (Fig. 1). The increasing trend is possibly related to economic support from government programs, since funding for innovative, sustainable, and ecological research is being considered to meet the demand for food and mitigate environmental pollution[30]. Figure 2 illustrates a co-citation map of authors collaborating in the field of Trichoderma formulation. The purpose of conducting this co-citation analysis is to visually portray the knowledge base of the specific area of review. The analysis identifies three distinct clusters, each represented by different colored nodes: blue, red, and green. The green cluster stands out as it is associated with Harman, who has the highest collaboration, working with nine researchers on Trichoderma formulation research. The red cluster, on the other hand, signifies the second-highest collaboration, led by Mukherjee et al.[31] with six researchers. Lastly, the pink cluster represents the lowest level of collaboration among researchers, with only two researchers working together in this area.

    Figure 2.  Co-citation analysis of cited authors as the unit of analysis in the field of Trichoderma formulation.

    Table 2 showcases the country–wise citation analysis, and the series presented here seems to have large variability in distribution. In terms of total citations of studies dedicated to Trichoderma formulations. Citations count of articles by country as a unit of analysis represents the popularity of a field of research in a particular region. India with 20 publications having 115 citations topped the list and, therefore, is the most impactful country contributing to the existing body of knowledge in the said domain followed by Italy with four publications having 69 citations and Brazil with three publications having 51 citations. From the viewpoint of the total number of publications, India holds first position, having 20 publications, followed by Italy having four publications.

    Table 2.  Leading countries contributing to the existing body of knowledge in the field of formulation of Trichoderma.
    Sl. no. Country No. of publication Citations
    1 Argentina 1 20
    2 Belgium 2 30
    3 Brazil 3 51
    4 China 2 82
    5 Croatia 1 30
    6 Finland 1 37
    7 India 20 115
    8 Italy 4 69
    9 New Zealand 2 21
    10 Portugal 1 67
    11 South korea 1 54
     | Show Table
    DownLoad: CSV

    The top researchers working in the field of Trichoderma formulation are presented in Table 2. The authors' citation count represents the recognition of their research work in a particular field of research. It is quite clear from the list of 13 author's citations that all the authors have at least 10 citations to their name based on the total citation count. Among the 13 authors Park et al.[32] has the highest number of citations of 57 followed by Herrera-Téllez et al.[33] having 47 and Hewedy et al.[34] having 44 citations. The analysis of bibliographic coupling in the Trichoderma formulation domain is represented in Fig. 3. This technique utilizes references from existing publications to elucidate the relevant literature[35]. For this study, five thematic clusters have been identified, labeled green, red, blue, yellow, and purple. Among these interconnected groups, India stands out as the country with the most extensive collaboration network, linked with 15 other countries. Given that India also holds the highest number of published documents (115), it was anticipated to be the central node in this cooperation network. The findings demonstrate the strong relationships between researchers and their respective institutional affiliations, emphasizing the scientific cooperation aimed at developing sustainable and ecologically sound strategies for crop protection in a competitive manner[36].

    Figure 3.  Bibliographic coupling of articles in the field of Trichoderma formulation.

    In Fig. 4, a co-occurrence analysis of keywords with a minimum threshold of five occurrences is displayed. The network illustrates the most frequently utilized terms within the 'Trichoderma formulation' research domain, capturing the essence of the article's core content. The prevalence of these keywords can be indicative of the research direction and content within this specific field[37]. This analysis allows for the identification of developmental trends within a field and a comprehensive understanding of its current research status[38]. The co-occurrence graph of keywords reveals a total of four co-occurrence clusters (Fig. 4), encompassing themes like biocontrol, formulation, Trichoderma, and shelf life. Each cluster is further examined below, providing an in-depth portrayal of the prominent topics within the Trichoderma formulations landscape during the research period. Annual publication number leading years contributing to the existing body of knowledge in the field of formulation of Trichoderma is presented in Table 3.

    Figure 4.  Co-occurrence analysis based upon keywords from articles in the field of Trichoderma formulation.
    Table 3.  Annual publication number leading years contributing to the existing body of knowledge in the field of formulation of Trichoderma.
    Sl. no. Year No. of publication
    1 2016 8
    2 2017 13
    3 2018 21
    4 2019 16
    5 2020 20
    6 2021 17
    7 2022 15
    8 2023 8
     | Show Table
    DownLoad: CSV

    The shift in annual publication counts serves as a vital benchmark for gauging the progress of a research field, lending insights into potential development trends[28]. Figure 4 provides a clear portrayal of the publication distribution in Trichoderma formulation from 2016 to 2023, illustrating a noticeable increase in annual article output. This surge suggests a heightened interest in the field over the past few years. Scientific research fields typically undergo a four-stage evolution[39] ; (1) the inception phase, characterized by the introduction of novel research areas or directions by notable scientists; (2) the expansion phase, where scientists gravitate toward the emerging research direction, leading to a proliferation of discussion topics; (3) the stabilization phase, marked by the amalgamation of new knowledge to form a distinct research context; and (4) the contraction phase, in which the number of new publications diminishes. Notably, the research on Trichoderma formulation seems to be currently in the expansion phase[40].

    The publication pattern reveals a growing research interest in Trichoderma formulations, a relatively new field that is attracting increasing enthusiasm among scholars. Notably, the top contributors to this area, as identified through VOS viewer analysis, include key individuals, organizations, sources, and countries. Park emerges as the leading author based on citation count, followed by Herrera-Téllez and Hewedy. China, Portugal, Italy, and South Korea are recognized as major contributors to research in this field, as reflected in their citation counts. Conversely, India leads in terms of document count, demonstrating a significant contribution to the literature.

    Co-citation and bibliographic coupling analyses have identified three distinct thematic clusters. In the co-citation analysis, these clusters relate to application methods, types of Trichoderma formulations, and their biocontrol efficacy. Additionally, insights from the bibliometric analysis of biopesticide formulations have facilitated the integration of methods and strategies aimed at enhancing the effectiveness of Trichoderma formulations.

    This paper conducts a bibliometric analysis to critically examine articles related to biological control, focusing specifically on those published in various indexed journals, and offers a comprehensive overview of the evolution of Trichoderma formulations over time. The primary objective of this research is to investigate and characterize the key literature on this topic, covering historical, current, and emerging developments in this dynamic field. Bibliometric techniques are employed to visualize the Trichoderma formulation landscape.

    To achieve this goal, the study analyses a dataset of articles obtained from the core collection databases of Scopus and Web of Science. Within the scope of this study, significant publications on Trichoderma formulations are meticulously reviewed to highlight the potential trajectory of Trichoderma's role in biological disease control in plants. The study identifies and examines the various developmental phases of Trichoderma formulations, offering a comprehensive analysis that can shape the future of this critical research area. Given the scarcity of comprehensive bibliometric studies on Trichoderma formulation research, this study seeks to fill this gap, making a significant contribution to the extensive readership interested in Trichoderma.

    This study has several limitations and challenges that must be considered by future researchers. First, the study relies on a single database, which could restrict the amount of available data. Additionally, the search criteria were limited to research articles, and only those with specific phrases in the title were included, which may not represent the complete dataset. However, Trichoderma's biological control mechanisms against plant fungal and nematode diseases involve various strategies, including competition, antibiosis, antagonism, and mycoparasitism. In addition to these, Trichoderma enhances plant growth and induces systemic resistance in plants, making it effective in controlling a wide range of plant fungal and nematode diseases[41]. Although biological control is effective, it generally requires time to become established in the environment, making it a slower process. Therefore, optimizing the formulation of Trichoderma-based products is essential to ensure their stability and efficacy. It is important to ensure that these formulations are compatible with other agricultural treatments, such as chemical fertilizers and pesticides, to maximize their overall effectiveness. Proper formulation can improve the shelf life, ease of application, and survival of Trichoderma under varying environmental conditions. Ongoing research is necessary to refine these formulations for broader application in integrated pest management programs[42]. This is a significant limitation as the analysis was restricted to articles published in journals, excluding other valuable sources like reviews, conferences, books, and book chapters. To overcome this limitation, future researchers should consider utilizing additional databases such as Scopus and Science Direct, which can provide more comprehensive data. This limitation may have impacted the study's ability to provide a comprehensive overview of the field.

    The authors confirm contribution to the paper as follows: conceptualization: Kumar V, Mishra KK, Panda SR; writing − original draft preparation: Kumar V, Wagh AK, Mishra KK; writing − review and editing: Panda SR, Kumar V, Wagh AK; supervision: Kumar V, Panda SR, All authors have read and agreed to the published version of the manuscript.

    The data that support the findings of this study are available on request from the corresponding authors.

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Xing A, Shu Z, Huang P, Zhang Y, Sui X, et al. 2024. Exogenous hydrogen sulfide enhanced Al stress tolerance in tea plant Camellia sinensis. Beverage Plant Research 4: e024 doi: 10.48130/bpr-0024-0013
    Xing A, Shu Z, Huang P, Zhang Y, Sui X, et al. 2024. Exogenous hydrogen sulfide enhanced Al stress tolerance in tea plant Camellia sinensis. Beverage Plant Research 4: e024 doi: 10.48130/bpr-0024-0013

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Exogenous hydrogen sulfide enhanced Al stress tolerance in tea plant Camellia sinensis

Beverage Plant Research  4 Article number: e024  (2024)  |  Cite this article

Abstract: Al is an essential element for the growth of tea plants roots, but excessive Al affects growth and development of Camellia sinensis. The underlying mechanism, particularly regulation of gas signaling molecule H2S, remains unclear. This study aims to uncover the function of H2S on C. sinensis under Al stress by treating hydroponic tea seedlings with different Al concentration, Na2S (H2S donor) and DL-propargylglycine (PAG, synthesis inhibitor). High concentration of Al inhibits growth of tea roots, while H2S significantly improves the effects caused by Al stress. Whether it is 2 mM Al3+ or 4 mM Al3+, H2S reduces content of Al in the entire plant and roots, increases root activity, further promotes root growth, increases fresh and dry weight, regulates ion homeostasis, improves cell structure, increases chlorophyll content, and thus reduces the damage of Al toxicity in C. sinensis. Moreover, in response to the stress of 2 mM Al3+, H2S simultaneously alleviates Al stress by regulating substances related to antioxidant pathways, increasing content of GSH and GSSG, enhancing activity of GST, GR, LCD, and key components of tea, in order to alleviate Al stress. These approaches have effectively improved Al tolerance of C. sinensis, providing a new perspective for the study of H2S enhancing Al tolerance.

    • Tea plant [Camellia sinensis (L.) O. Kuntze], is suitable for growing in acidic soil with pH 4.5−6.5. Aluminum (Al) toxicity is currently a crucial factor limiting plant growth in acidic environments, because when the pH of the soil is less than 5, Al can be transformed into phytotoxic trivalent cation (Al3+) that are readily absorbed by plants, thereby affecting plant growth[1]. As an Al hyperaccumulating plant, C. sinensis can contain up to 30,000 mg·kg−1 of Al in its mature leaves without showing symptoms of Al toxicity[2]. Appropriate Al concentration promotes the growth and development of tea plants. Once it exceeds 1 mmol·L−1, C. sinensis suffer from a negative effect on its normal growth[3].

      Various strategies for plants to cope with Al toxicity include external exclusion mechanisms such as increasing Al chelation and reducing Al uptake by plants, as well as increasing antioxidant enzyme activity and reducing toxic substances caused by reactive oxygen species and free radicals, among other internal detoxification mechanisms[4]. Meanwhile, excessive Al also has a certain impact on the tea quality components of tea polyphenols, catechins, amino acids, caffeine and other substances[5]. Not only that, tea consumption also increases dietary intake of Al, which is thought to be associated with Alzheimer's disease[6]. Therefore, exploration of measures to reduce content of Al in C. sinensis is of great significance in alleviating Al stress and improving tea quality.

      Hydrogen sulfide (H2S) is regarded as a poisonous gas and atmospheric pollutant, but it was subsequently found to be the third gaseous signaling molecule after nitric oxide (NO) and carbon monoxide (CO)[7]. And synthesizes endogenous H2S mainly through L-cysteine desulphydrase (LCD), which is widely present in plants[8]. Recently, research on H2S has begun to reveal the role of these molecules in regulating plant abiotic and biotic stress resistance responses. Through the exogenous application of H2S donors, H2S has been proven to regulate plant growth and increase plant tolerance to drought, salt, temperature, and metal stress. It can be seen that H2S plays vital roles in facilitating plant with tolerance to environmental stresses[9]. However, the role of H2S in alleviating Al stress of C. sinensis is still unclear.

      There are many studies on Al enrichment in tea plants, but currently there is a lack of research on H2S signaling molecules for Al tolerance in tea plants. Now, through different hydroponic treatments (0.4Al, H2S + 0.4Al, PAG + 0.4Al, 2Al, H2S + 2Al, PAG + 2Al, 4Al, H2S + 4Al, PAG + 4Al), we investigated the effects of H2S preapplication on the biomass, the content and transfer rate of Al and other elements in different tissues, the content of chlorophyll, photosynthetic indexes, the ultrastructure, the antioxidant enzyme activity and tea quality components under Al stress. Preliminary exploration of the role of exogenous H2S in the physiological response of tea plants to Al stress provides new ideas for further research on alleviating Al stress and reducing Al accumulation in tea plants.

    • For the experiment, annual cutting seedlings of C. sinensis cv. 'Zhongcha 108' were obtained from the Nanjing (Ya Run Tea Co., Ltd., Jiangsu Province, China). The tea seedlings were firstly pre-cultured in water for 5 d, then transferred to 1/8, 1/4, and 1/2 total nutrient solutions to culture for 5 d in each strength nutrient solution, and finally transferred to total nutrient solutions for 10 d (culture medium was replaced every 5 d)[10]. The seedlings with consistent growth were used to carry out the subsequent treatment assays with H2S or PAG and Al3+ as shown in Table 1. For treatments, Al2(SO4)3·18H2O, Na2S·9H2O and DL-propargylglycine (PAG) were the Al3+ donor[11], H2S donor[12] and L-cysteine desulfurase (LCD) inhibitor[13], respectively. And solution pH was adjusted to 4.5 ± 0.1 with 1.0 mol·L−1 NaOH or 1.0 mol·L−1 HCl. The experiments were performed in the Intelligent Greenhouse of Nanjing Agricultural University (China), controlled growth room at 25 °C/22 °C with 16 h light/8 h dark cycle, 30,000 l x light intensity and a relative humidity of 75%.

      Table 1.  Description of nine experimental treatments.

      Treatments Days 1−15 Days 15−30
      0.4Al (control) 0.4 mmol·L−1 Al3+ 0.4 mmol·L−1 Al3+
      H2S + 0.4Al 100 μmol·L−1 H2S + 0.4 mmol·L−1 Al3+ 0.4 mmol·L−1 Al3+
      PAG + 0.4Al 1 mmol·L−1 PAG + 0.4 mmol·L−1 Al3+ 0.4 mmol·L−1 Al3+
      2Al 0.4 mmol·L−1 Al3+ 2 mmol·L−1 Al3+
      H2S + 2Al 100 μmol·L−1 H2S + 0.4 mmol·L−1 Al3+ 2 mmol·L−1 Al3+
      PAG + 2Al 1 mmol·L−1 PAG + 0.4 mmol·L−1 Al3+ 2 mmol·L−1 Al3+
      4Al 0.4 mmol·L−1 Al3+ 4 mmol·L−1 Al3+
      H2S + 4Al 100 μmol·L−1 H2S + 0.4 mmol·L−1 Al3+ 4 mmol·L−1 Al3+
      PAG + 4Al 1 mmol·L−1 PAG + 0.4 mmol·L−1 Al3+ 4 mmol·L−1 Al3+
    • Plants were collected and separated into young leaf (the first and second leaf from the top of plants), mature leaf (remaining leaves), stem and root. Fresh weight (FW) of seedlings were weighed instantly after harvesting and then placed into an oven at 105 °C for 30 min and then baked at 80 °C until biomass became stable. The dry weight (DW) immediately weighed after removal from the oven.

    • Root activity was measured using the 2,3,5-tripheyl tetrazolium chloride (TTC) method[14]. About 0.5 g of fresh root tips were placed in a mixture of 5 mL 1% TTC and 5 mL phosphate buffer for 1 h at 37 °C in the dark. The assays were terminated by adding 2 mL 1.0 mol·L−1 H2SO4 to the reaction mixture. The reduced TTC was extracted with 3-4 mL ethyl acetate, then ethyl acetate was added to the 10 mL level, and absorbance was read at 485 nm.

    • Leaf fragments without veins were collected from randomly selected plants, then fixed 24 h in 2.5 % glutaraldehyde solution and stored at 4 °C. Samples were rinsed three times with the same phosphate-buffered saline (PBS, pH 7.2), and post-fixed in 1% osmium oxide for 1 h, washed three times with distilled water. The samples were dehydrated in a graded series of ethanol (50%, 70%, 80%, and 100 %) and at the end treated with absolute acetone for 24 h. Ultra-thin sections (≤ 100 nm) of specimens were prepared for viewing.

    • Chlorophyll a and chlorophyll b of randomly selected mature leaves per treatment were measured as described previously[15]. Samples were completely immersed with 10.0 mL mixture of acetone-95% ethanol-water (9:9:2, v:v) and transferred into tubes placed in a dark place until the leaves turn completely white. The OD665 and OD649 values were used to calculate chlorophyll content. A LI-6400XT portable photosynthesis system (Li-Cor Biosciences, Lincoln, Nebraska, USA) was used to measure net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) with 1,200 μmol·m−2·s−1 illuminance and 500 μmol·mol–1 flow rate.

    • The plant samples with 0.2 g were placed into the digestion vessels, mixed with HNO3 : HClO4, (4:1, v:v) and digested in microwave digestion system. The concentrations of Al, calcium (Ca), magnesium (Mg), manganese (Mn), iron (Fe) and zinc (Zn) in the filtrate were determined using inductively coupled plasma optical emission (ICP-OES, PerkinElmer Inc.) following a standard procedure.

    • Lipid peroxidation was measured in terms of malondialdehyde (MDA) content according to Alatawi et al.[16]. Fresh leaves (0.1 g) were ground and extracted in 1 mL of 10% trichloroacetic acid (TCA), then the supernatant was collected by centrifuging at 5,000 rpm for 10 min. 0.5 mL supernatant (0.5 mL distilled water as control) were homogenized in 0.5 ml of 0.6 % 2-thiobarbituric acid (TBA) and heat in boiling water for 15 min, then cooled until room temperature. The absorbance of the supernatant was measured at 532, 600, and 450 nm.

      Proline content was determined using the acid ninhydrin method[17]. First, 0.1 g of leaf samples was added to 1 ml of 3% sulfosalicylic acid solution and extracted in boiling water for 10 min, then centrifuged at 5,000 rpm for 10 min. Next, 0.2 mL of supernatant was homogenized and mixed with 0.2 mL of acetic acid and 0.2 mL of 2.5% acid ninhydrin and kept at boiling point for 30 min, then cooled until room temperature, 0.4 mL of toluene treated and then oscillated by vortex for 30 s. After 10 min, supernatant centrifuged at 3,000 rpm for 5 min. Finally, the absorbance was scored at 520 nm.

    • The glutathione (GSH) and oxidized glutathione (GSSG) were measured by GSH and GSSG kit (NO. BC1170, NO. BC1180; Beijing Solarbio Science & Technology Co., Ltd., China). LCD enzyme was detected by referring to the LCD kit (NO. MBE21193; Nanjing Maibo Biotechnology Co., Ltd., China). The activities of glutathiones-transferase (GST) and glutathione reductase (GR) was determined following the description by kit (NO. BC0350, NO. BC1160; Beijing Solarbio Science & Technology Co., Ltd., China). Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) was performed according to instructions of the kits (NO. R22262, NO. R3031, NO. R22072; Shanghai Yuanye Biotechnology Co., Ltd., China), respectively.

    • The contents assay viz. tea polyphenols, catechins, amino acids and caffeine was measured according to GB/T 8313-2018[18], GB/T 8314-2013[19], and GB/T 8312-2013[20], respectively.

    • All the data were from three independent experiments with three biological repeats. The experimental data were statistically processed using Excel 2016, GraphPad Prism 8.0 and variance analysis software SPSS 20.0 (SPSS Inc. version 22.0, Chicago, IL, USA). Different lowercase letters on the graphs indicate that the mean values among different H2S conditions under the same Al concentration treatment were statistically different at p < 0.05 level, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition at p < 0.05 level.

    • As expected, new root of C. sinensis treated with 2Al and 4Al was less than that of normal 0.4Al culture, but early application of H2S compared to lone Al treatment effectively promoted the root development, while PAG + Al significantly inhibited root growth (Fig. 1). Moreover, application of PAG not only inhibited normal development of root system, but also inhibited the growth of leaves (Fig. 1c, f & i). Chlorosis, even leaf abscission symptoms in leaves have also occurred (Fig. 1c, f & i).

      Figure 1. 

      Effect of different treatments on symptoms, (a) 0.4Al, (b) H2S + 0.4Al, (c) PAG + 0.4Al, (d) 2Al, (e) H2S + 2Al, (f) PAG + 2Al, (g) 4Al, (h) H2S + 4Al, (i) PAG + 4Al, and (j) root activity in C. sinensis. Different lowercase letters in (j) represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

      To further clarify whether H2S is beneficial for tea root growth under different Al conditions, we explored root activity. We observed higher concentrations (2Al and 4Al) resulted in a greatly decrease in root activity (Fig. 1j). And an increase of 37.59%, 58.42%, and 19.55% in root activity under H2S pretreatment compared to the separate 0.4Al, 2Al and 4Al treatments, respectively (Fig. 1j). However, exogenous PAG treatment significantly inhibited root activity compared to various Al concentrations (Fig. 1j).

    • Overall, the total fresh weight (FW) and dry weight (DW) of tea plants were both increased by early application of H2S, while the use of PAG reduced the FW and DW of C. sinensis (Fig. 2e, j). Moreover, the results showed that, except for H2S + 4Al, which did not increase FW in the leaves compared to 4Al, the FW of other different tissues under H2S + Al treatments showed an increase in FW compared to the single Al treatment (Fig. 2ad). In addition, the DW of other tissues increased under H2S + Al treatments compared to single Al treatment for tea seedlings, except for H2S + 4Al which showed decrease in DW of old leaves compared to 4Al (Fig. 2fi).

      Figure 2. 

      Fresh and dry weight in (a, f) young leaves, (b, g) matures leaves, (c, h) stems, (d, i) roots, and (e, j) total content of Al of C. sinensis cultured with different treatments. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

    • There was no significant decrease in content of Al between pre-applied H2S treatment and single Al treatment in young leaves (Table 2). Nevertheless, compared with 0.4Al treatment, content of Al in roots markedly increased when H2S was applied in advance, while accumulation of Al in roots was dramatically reduced when H2S was applied in advance to the 2 mM Al3+ and 4 mM Al3+ treatments (Table 2). Meanwhile, compared to other treatments within the group, content of Al was the highest in roots when PAG-pretreated was applied in advance, with similar performance in total Al content (Table 2). Unusually, pretreatment with H2S increased content of Al in mature leaves compared to Al treatment alone, and there was a similar trend of Al accumulation in stems (Table 2). Under normal Al concentration, the translocation factor (TF) of Al of 0.4Al is the highest, which is 1.7 times that of H2S + 0.4Al and 10.625 times that of PAG + 0.4Al (Table 2). Whereas, TF of Al demonstrated H2S + Al > Al > PAG + Al after 2Al and 4Al treatment (Table 2).

      Table 2.  Effects on content and translocation factor (TF) of Al in C. sinensis under different treatments.

      Elements Treatment YL (mg·kg−1) ML (mg·kg−1) S (mg·kg−1) R (mg·kg−1) Total content (mg·kg−1) TF (%)
      Al 0.4Al 551.30 ± 25.52Ba 1204.96 ± 324.00Aa 427.13 ± 12.20Ba 2566.89 ± 96.52Bc 4750.29 ± 207.35Bc 0.85 ± 0.14Aa
      0.4Al + H2S 474.49 ± 57.74Ba 1605.65 ± 30.85Ba 558.06 ± 69.96Ca 5323.33 ± 506.90Cb 7961.52 ± 573.30Bb 0.50 ± 0.04Bb
      0.4Al + PAG 291.67 ± 33.05Cb 1125.82 ± 345.62Aa 537.35 ± 118.21Aa 23523.28 ± 1412.99Ba 25478.12 ± 1623.32Ba 0.08 ± 0.01Bc
      2Al 700.68 ± 14.51Ab 1232.58 ± 102.65Ab 735.12 ± 40.15Ab 15651.00 ± 387.50Ab 18319.37 ± 478.25Ab 0.17 ± 0.01Bb
      2Al + H2S 758.33 ± 51.39Ab 2000.13 ± 209.09Aa 976.59 ± 29.70Aa 8574.85 ± 700.31Bc 12309.90 ± 638.39Ac 0.44 ± 0.05Ba
      2Al + PAG 999.06 ± 45.47Ba 1101.78 ± 48.02Ab 669.60 ± 131.31Ab 28361.41 ± 199.73Aa 31131.86 ± 296.48Aa 0.10 ± 0.01ABc
      4Al 771.52 ± 123.22Ab 1342.59 ± 60.73Aa 675.39 ± 120.29Aa 14741.91 ± 2122.85Ab 17531.41 ± 2218.98Ab 0.19 ± 0.03Bb
      4Al + H2S 819.06 ± 20.35Ab 1475.13 ± 107.29Ba 732.93 ± 62.82Ba 10066.06 ± 835.49Ac 13093.18 ± 954.89Ab 0.30 ± 0.02Aa
      4Al + PAG 1285.44 ± 106.37Aa 1086.46 ± 135.20Ab 756.13 ± 104.12Aa 26682.34 ± 3130.59ABc 29810.37 ± 3150.15Aa 0.12 ± 0.02AAc
      Values are the mean ± SD (n = 3). Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.
    • Content of Ca increased in the solution with H2S or PAG pre-applied, and this increase was more elevated in 0.4Al than 2Al, 4Al in young leaves, while more increased in 2Al and 4Al than 0.4Al in mature leaves (Table 3). In stems, application of PAG remarkably enhanced the concentrations of Ca under 0.4Al, but decreased content of Ca in 2Al and 4Al (Table 3). Moreover, results in roots showed that content of Ca under H2S + 0.4Al was 2.71 times that of 0.4 Al, and content of Ca in PAG + 0.4Al was 4.5 times that of 0.4 Al, but H2S + 4Al and PAG + 4Al inhibited content of Ca compared to 4Al, and changes in content of Ca between 2Al, H2S + 2Al, and PAG + 2Al groups were relatively small (Table 3). After H2S combined 2Al significantly improved content of total Ca, while a little effect on 0.4Al and 4Al (Table 3). In addition, the TF of Ca exhibited 0.4Al > H2S + 0.4Al > PAG + 0.4Al, while H2S +2 Al and PAG + 2Al have no significant effect on the TF of Ca compared to 2Al, only H2S + 4Al significantly promoted TF of Ca compared to 4Al (Table 3).

      Table 3.  Effects on content and translocation factor (TF) of Ca, Mg, Zn, Mn, Fe in C. sinensis under different treatments.

      Elements Treatment YL (mg·kg−1) ML (mg·kg−1) S (mg·kg−1) R (mg·kg−1) Total content (mg·kg−1) TF (%)
      Ca 0.4Al 6168.53 ± 606.30Bb 13894.03 ± 340.79Aab 2746.77 ± 24.59Bb 476.37 ± 41.02Cc 23285.69 ± 1696.58Bb 47.92 ± 0.76Aa
      0.4Al + H2S 8064.69 ± 66.16Ca 13027.66 ± 1043.18Bb 2781.84 ± 61.89Cb 1289.49 ± 60.48Bb 25163.67 ± 436.51Cb 18.53 ± 0.58Ab
      0.4Al + PAG 9322.85 ± 1413.64Aa 14978.35 ± 230.29Aa 3606.75 ± 523.74Aa 2144.36 ± 58.02Aa 30052.32 ± 1938.42Aa 13.03 ± 1.24Ac
      2Al 11223.02 ± 223.36Aa 12447.40 ± 1926.26Ab 3559.11 ± 198.46Aa 1573.00 ± 74.02Ba 28802.53 ± 598.30Ac 17.33 ± 0.62Ba
      2Al + H2S 11693.96 ± 749.31Aa 15968.96 ± 553.09Aa 3955.32 ± 217.77Aa 1695.65 ± 273.89Aa 33313.87 ± 1007.07Aa 19.05 ± 3.82Aa
      2Al + PAG 10558.09 ± 739.25Aa 15748.44 ± 629.97Ba 3026.63 ± 321.80Ab 1905.82 ± 193.23ABa 31238.99 ± 388.96Ab 15.51 ± 1.78Aa
      4Al 10986.74 ± 1426.59Aa 12852.33 ± 968.51Ab 3355.19 ± 272.64Aab 1878.07 ± 191.42Aa 29072.34 ± 1204.10Aa 14.57 ± 1.35Cb
      4Al + H2S 10405.55 ± 102.64Ba 14656.58 ± 769.17ABa 3566.27 ± 65.69Ba 1511.59 ± 84.73ABb 30140.00 ± 1162.14Ba 18.96 ± 0.69Aa
      4Al + PAG 10806.80 ± 1205.83Aa 12881.85 ± 15.75Bb 3227.25 ± 1.88Ab 1713.41 ± 164.33Bab 28629.31 ± 1365.33Aa 15.77 ± 0.97Ab
      Mg 0.4Al 2161.53 ± 297.57Ba 2987.80 ± 291.75Aab 1388.72 ± 43.03Bb 346.52 ± 15.37Bc 6884.56 ± 597.72Bb 18.87 ± 1.45Aa
      0.4Al + H2S 2679.11 ± 76.29Ca 2869.31 ± 12.76Ab 1326.99 ± 29.76Bb 1384.29 ± 37.80Aa 8259.70 ± 93.16Ca 4.97 ± 0.21Bc
      0.4Al + PAG 2551.51 ± 378.60Ba 3266.05 ± 76.24Aa 2152.37 ± 144.18Aa 497.68 ± 22.78Ab 8467.61 ± 526.12ABa 16.04 ± 1.39Ab
      2Al 3203.55 ± 83.10Aa 2612.19 ± 216.71Ab 1578.27 ± 51.43Ab 924.90 ± 98.81Ab 8318.92 ± 253.97Ac 8.06 ± 0.95Bb
      2Al + H2S 3307.60 ± 162.06Aa 3142.00 ± 338.93Aa 1660.00 ± 103.48Ab 1322.66 ± 58.80Aa 9432.27 ± 159.11Aa 6.14 ± 0.43Ab
      2Al + PAG 3089.27 ± 163.42Aa 3290.93 ± 108.91Aa 1908.25 ± 31.93Ba 498.33 ± 39.52Ac 8786.78 ± 71.55Ab 16.70 ± 1.37Aa
      4Al 3610.66 ± 446.66Aa 2809.35 ± 171.88Aa 1442.35 ± 119.96ABb 954.41 ± 122.46Ab 8816.77 ± 562.41Aa 8.30 ± 0.78Bb
      4Al + H2S 3059.80 ± 57.45Bb 2752.72 ± 222.97Aa 1561.74 ± 53.55Ab 1284.43 ± 54.96Aa 8658.69 ± 280.14Ba 5.74 ± 0.13Ab
      4Al + PAG 2707.61 ± 5.02ABb 2584.47 ± 406.13Ba 2072.19 ± 16.34ABa 387.73 ± 33.22Bc 7752.01 ± 387.21Bb 19.12 ± 2.39Aa
      Zn 0.4Al 8.70 ± 1.12Ca 12.22 ± 1.13Ba 18.92 ± 1.49Bb 23.29 ± 1.72Bc 63.12 ± 2.34Bc 1.72 ± 0.14Aa
      0.4Al + H2S 11.58 ± 3.00Ca 14.66 ± 2.74Aa 20.42 ± 2.36Bb 82.47 ± 4.76Ca 129.13 ± 5.16Ba 0.57 ± 0.04Bc
      0.4Al + PAG 9.33 ± 1.22Ca 12.43 ± 0.50Ba 25.11 ± 0.62Ba 59.10 ± 2.76Aa 105.96 ± 1.00Cb 0.80 ± 0.07Cb
      2Al 13.68 ± 1.25Bab 14.98 ± 1.59ABa 27.60 ± 2.46Aa 74.61 ± 5.32Aa 130.86 ± 3.45Ab 0.76 ± 0.11Bb
      2Al + H2S 15.49 ± 0.95Ba 16.72 ± 1.98Aa 26.31 ± 3.59ABa 123.25 ± 4.99Aa 181.77 ± 5.41Aa 0.48 ± 0.02Cc
      2Al + PAG 11.80 ± 0.93Bb 20.59 ± 4.06Aa 30.27 ± 1.78Aa 55.57 ± 2.46Aa 118.24 ± 3.15Bc 1.13 ± 0.07Aa
      4Al 17.09 ± 2.08Ab 18.44 ± 4.07Aa 29.14 ± 2.30Aa 82.54 ± 7.89Aa 147.20 ± 14.19Ab 0.78 ± 0.09Ba
      4Al + H2S 21.37 ± 0.92Aa 17.90 ± 0.30Aa 31.47 ± 5.39Aa 101.06 ± 5.07Aa 171.80 ± 9.69Aa 0.70 ± 0.02Aa
      4Al + PAG 14.61 ± 1.07Ab 13.16 ± 0.38Bb 33.98 ± 3.70Aa 67.89 ± 9.40Aa 129.63 ± 7.39Ab 0.93 ± 0.19ABa
      Mn 0.4Al 434.54 ± 49.93Bb 545.22 ± 57.44Ab 166.17 ± 11.21Ab 134.39 ± 12.51Cc 1280.32 ± 34.67Cc 8.60 ± 1.20Aa
      0.4Al + H2S 331.68 ± 11.06Cb 877.57 ± 9.52Aa 344.66 ± 11.87Ba 754.57 ± 31.78Ab 2308.49 ± 11.48Bb 2.06 ± 0.12Bb
      0.4Al + PAG 834.44 ± 114.52Aa 550.37 ± 95.76Ab 133.36 ± 16.66Ac 4346.17 ± 226.23ABa 5864.34 ± 188.71Aa 0.35 ± 0.03Bc
      2Al 580.86 ± 12.29Ac 493.94 ± 29.55Ac 140.13 ± 9.00Bb 331.60 ± 27.71Bc 1546.53 ± 53.41Bc 3.68 ± 0.26Ba
      2Al + H2S 725.34 ± 47.40Ab 812.18 ± 82.33Aa 441.12 ± 40.94Aa 700.79 ± 23.40Bb 2679.43 ± 33.48Ab 2.83 ± 0.08Ab
      2Al + PAG 928.92 ± 45.21Aa 624.51 ± 22.29Ab 98.97 ± 8.63Ab 3967.98 ± 23.31Ba 5620.38 ± 56.40Aa 0.42 ± 0.01Ac
      4Al 460.62 ± 66.33Bb 555.60 ± 64.32Ac 177.93 ± 12.21Ab 556.44 ± 62.27Ab 1750.59 ± 105.01Ab 2.16 ± 0.17Cb
      4Al + H2S 628.13 ± 14.12Ba 689.53 ± 39.43Ba 276.52 ± 2.49Ca 555.42 ± 21.57Cb 2149.60 ± 47.85Cb 2.87 ± 0.16Aa
      4Al + PAG 435.88 ± 72.96Bb 601.91 ± 11.98Aab 107.04 ± 23.12Ac 4802.02 ± 610.68Aa 5946.86 ± 651.79Aa 0.24 ± 0.02Cc
      Fe 0.4Al 83.08 ± 8.24Bc 228.98 ± 29.41Ab 116.78 ± 11.68Ab 212.67 ± 18.38Cc 641.51 ± 8.04Bc 2.03 ± 0.29Aa
      0.4Al + H2S 142.21 ± 15.79Aa 399.33 ± 0.52Aa 292.37 ± 11.60Aa 401.46 ± 66.03Ab 1235.37 ± 78.04Ab 2.11 ± 0.34Aa
      0.4Al + PAG 111.92 ± 8.22Bb 230.35 ± 33.40ABb 110.94 ± 14.25Ab 956.68 ± 72.57Aa 1409.89 ± 74.53Ba 0.48 ± 0.04Ab
      2Al 147.45 ± 5.50Ab 207.06 ± 16.44Aa 122.89 ± 29.28Aa 702.60 ± 109.64Ab 1180.00 ± 150.22Ab 0.69 ± 0.07Ba
      2Al + H2S 130.75 ± 8.31Ab 257.56 ± 31.45Ba 136.29 ± 7.22Ca 521.81 ± 202.62Ab 1046.41 ± 198.89Ab 1.13 ± 0.50Ba
      2Al + PAG 216.42 ± 11.66Aa 266.43 ± 43.53Aa 110.46 ± 8.78Aa 1110.40 ± 18.95Aa 1703.70 ± 19.91Aa 0.53 ± 0.04Aa
      4Al 134.64 ± 13.60Ab 208.35 ± 12.59Ab 105.13 ± 5.55Ab 534.53 ± 88.15Bb 982.65 ± 96.52Ab 0.85 ± 0.16Bb
      4Al + H2S 145.29 ± 2.86Ab 270.95 ± 21.28Ba 201.43 ± 16.05Ba 368.04 ± 37.29Ab 985.70 ± 44.68Ab 1.69 ± 0.16ABa
      4Al + PAG 230.61 ± 38.35Aa 163.31 ± 45.36Bb 103.44 ± 3.47Ab 944.38 ± 142.73Aa 1441.74 ± 137.29Ba 0.54 ± 0.14Ac
      Values are the mean ± SD (n = 3). Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

      Content of Mg in young leaves were found to significantly inhibited only in H2S + 4Al and PAG + 4Al compared to 4Al, but there was no significant change in content of Mg between treatments at only 4Al in mature leaves (Table 3). The application of exogenous PAG contributed to increase in content of Mg in stems, but H2S had a small effect on the level of Mg compared to Al alone in stems (Table 3). However, H2S significantly increased Mg levels in roots (Table 3). It was found that the change in total content of Mg was not significant under H2S + 4Al compared to 4Al, while content of Mg under other H2S + Al treatments significantly increased compared to Al alone (Table 3). However, the TF of Mg was inhibited by 73.66% under H2S + 0.4Al compared to 0.4Al, 23.82% under H2S + 2Al compared to 2Al, and 30.84% under H2S + 4Al compared to 4Al (Table 3).

      It was demonstrated that H2S promoted an increase in content of Zn in different tissues (Table 3). Meanwhile, the total content of Zn also showed that H2S-pretreated significantly promoted the accumulation of Zn in C.sinensis. The effects of H2S and PAG on TF of Zn under different Al concentrations were also inconsistent. Significantly inhibited TF of Zn was observed in exogenous H2S or PAG followed by 0.4Al, however, TF of Zn showed significant performance as PAG + 2Al > 2Al > H2S + 2Al, but there was no significant difference in the effect of exogenous H2S or PAG on TF of Zn at 4Al (Table 3).

      Content of Mn further increased after applying H2S + 2Al and H2S + 4Al to young leaves, while content of Mn was decreased but not significant in H2S + 0.4 Al compared with simple Al treatment (Table 3). It was H2S + Al that dramatically increased Mn levels compared to Al in mature leaves, consistent with the performance in stems (Table 3). In roots, it was PAG + Al that showed a significant increase in content of Mn compared to Al, and H2S significantly promoted an increase in Mn only at 0.4Al and 2Al (Table 3). It is interesting to note that total content of Mn was similar to the content of Mn under each treatment in roots (Table 3). The early application of PAG increased the accumulation of total Mn in tea plants compared to Al alone, but its TF was significantly inhibited, with TF of Mn at 0.4Al was 24.57 times that of H2S + 0.4Al, 2Al was 8.76 times that of H2S + 2Al, and 4Al was nine times that of H2S + 4Al.

      Exogenous H2S followed by 0.4Al resulted in a remarkable increase in the content of Fe in young leaves, but the effect of H2S on content of Fe was not significant at 2Al and 4Al, whereas PAG showed a significant increase in content of Fe (Table 3). H2S also increased content of Fe in mature leaves at various Al concentrations, as well as in stems (Table 3). Whereas, exogenous PAG significantly increased content of Fe in roots, and the total content of Fe was also significantly affected by exogenous PAG (Table 3). Under different Al concentrations, H2S + Al exhibited a promotion of Fe-TF, while PAG + Al inhibited TF of Fe (Table 3).

    • A clear cell membrane could be seen in normal Al concentration, and the well-developed chloroplast having regular arrangements of thylakoid membranes could also be observed (Fig. 3a). At the same time, no osmiophilic granules (OG) were present in chloroplasts under H2S + 0.4Al (Fig. 3b), but application of PAG + 0.4Al led to appearance of OG in chloroplasts (Fig. 3c). Under stress of 2Al, the chloroplast membranes (PE) were still visible, but OG appeared (Fig. 3d), H2S reduced OG (Fig. 3e), and the early application of PAG generated more OG (Fig. 3f). Although, chloroplast structure was relatively intact, cells with scattered stromal lamellae under 4Al stress, and the stromal lamellar structure of H2S + 4Al loosened even more (Fig. 3g, h), with solubilization and even vacuolation occurring in PAG + 4Al (Fig. 3i).

      Figure 3. 

      Different changes in ultrastructure of C. sinensis after different treatments. (a) 0.4Al, (b) H2S + 0.4Al, (c) PAG + 0.4Al, (d) 2Al, (e) H2S + 2Al, (f) PAG + 2Al, (g) 4Al, h: H2S + 4Al, (i) PAG + 4Al. PE: chloroplast membrane, Ch: chloroplast, SG: starch granules, Th: matrix lamellae, OG: osmiophilic granule. Scale bar = 1.0 μm.

    • An increase was observed in chl a content under H2S as compared to Al treatment alone, however, reduction of chl a showed in exogenous PAG, and chl b content has the same performance (Fig. 4a, b). Furthermore, total chlorophyll content also has the same trend, and with the increase of Al concentration, the total chlorophyll content of H2S + Al increases by 21.15%, 11.59%, and 17.64% compared to Al, respectively (Fig. 4c). Nevertheless, the results of chl a/chl b showed the opposite, namely PAG + Al > Al > H2S + Al (Fig. 4d).

      Figure 4. 

      Changes in chlorophyll content of C. sinensis after different treatments. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

      Pn under 0.4Al was significantly promoted by application of H2S, but pretreatment with PAG significantly decreased Pn (Fig. 5a). Differently, the effect of applying H2S and PAG on Pn showed an opposite trend at 2Al, and exogenous application of H2S and PAG showed significant inhibition compared to 4Al alone (Fig. 5a). Gs showed a consistent trend at 0.4Al and 4Al, with H2S + Al > Al > PAG + Al (Fig. 5b). Ci were different under different treatments with different Al concentrations, namely H2S + 0.4Al > 0.4Al > PAG + 0.4Al, 2Al > H2S + 2Al > PAG + 2Al, and PAG + 4Al > 4Al > H2S + 4Al (Fig. 5c). The results of Tr under normal Al concentration were consistent with those of Pn, Gs and Ci, but at high concentrations of Al, they showed 2Al > PAG + 2Al > H2S + 2Al, 4Al > H2S + 4Al > PAG + 4Al, respectively (Fig. 5d). Tr agreed with Pn, Gs, Ci results at normal Al concentration, but exhibited 2Al > PAG + 2Al > H2S + 2Al and 4Al > H2S + 4Al > PAG + 4Al, respectively, at Al stress concentrations (Fig. 5).

      Figure 5. 

      Changes in photosynthetic parameters of C. sinensis under different treatments. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

    • Interestingly, MDA content in leaves of H2S pretreatment was inhibited by 3.61% compared to 2Al, whereas preincubation of PAG significantly increased MDA content (Fig. 6a). Proline content significantly accumulated in Al stress compared to 0.4Al, and its content increases by 2.82% under H2S + 2Al compared to 2Al, while pretreatment with H2S before 4Al treatment did not inhibit lipid peroxidation through proline content (Fig. 6b).

      Figure 6. 

      The effect of different treatments on (a) MDA and (b) proline content in tea leaves. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

      Similar tendency was observed in roots and leaves under normal Al, with H2S + 0.4Al compared to 0.4Al not significantly increasing CAT activity by 15% and 16.67%, respectively (Fig. 7a & b). CAT showed the highest activity of H2S + 2Al in leaves, but the lowest activity in roots under H2S + 2Al (Fig. 7a & b). And CAT activity of leaves at 4Al was higher than that of H2S + 4Al at 4Al and PAG + 4Al, while the CAT activity in roots treated with PAG + 4Al was higher than that of 4Al and H2S + 4Al (Fig. 7a & b).

      Figure 7. 

      C. sinensis on antioxidant enzyme activities in (a), (c), (e) leaves and (b), (d), (f) roots with different treatments. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

      Similarly, POD activity showed the same trend in roots and leaves only under normal Al, with POD activity in PAG + 0.4Al greater than that in 0.4Al and H2S + 0.4Al (Fig. 7c & d). Meanwhile, it is noteworthy that POD activity after H2S + 2Al is 3.56 times compared to 2Al in leaves, while the lowest POD activity was observed in the roots at H2S + 2Al, and the same was showed PAG + Al > Al > H2S + Al at 4 Al (Fig. 7c & d). However, there was no significant difference between the treatments at 4Al for the leaves (Fig. 7c).

      Compared with 0.4Al treatment, H2S + 0.4Al treatment increased SOD activity in leaves and roots (Fig. 7e & f). However, there was no significant difference in SOD activity after applying H2S at 4 mM Al3+ in the roots and leaves (Fig. 7e & f).

      But the application of H2S and PAG under 2Al conditions in leaves failed to stimulate the activity of SOD, and pretreatment with H2S or PAG in roots dramatically decreased the activity of SOD (Fig. 7e & f). Furthermore, there was no significant difference in SOD activity among different treatments at 4Al in leaves and roots (Fig. 7e & f).

      GSH content in leaves under normal Al and 2Al all exhibited H2S + Al > Al > PAG + Al, but GSH exhibited Al > H2S + Al > PAG + Al in 4Al (Fig. 8a). The content of GSSG was decreased in PAG-treated at 0.4Al and 2Al, but was increased in PAG + 4Al cultures (Fig. 8b). Noteworthy, no significant change of GSH/GSSG was discovered when H2S or PAG was added together with Al treatment (Fig. 8c).

      Figure 8. 

      Effect of different treatments on (a) GSH content, (b) GSSG content, (c) GSH/GSSG, (d) GST activity and (e) GR activity in tea leaves. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

      It was found that GST activity in leaves was higher in H2S + 0.4Al than under 0.4Al and PAG + 0 .4Al treatments (Fig. 8d). And GST activity exhibited the highest in H2S + Al, followed by 2Al, and the lowest in PAG + 2Al. Unlike under 4Al where the activity of GST was inhibited by 4Al treatment with H2S and PAG, although the level of decrease was not significant (Fig. 8d).

      Tea leaves exposed to H2S + 0.4Al treatment exhibited a significant increase of GR activity in comparison with 0.4Al alone and PAG + 0.4Al samples (Fig. 8e). PAG + 2Al and H2S + 4Al treatments had the lowest GR activity compared with 2Al and 4Al, respectively (Fig. 8e).

      LCD activity only showed H2S + 0.4Al > 0.4Al > PAG + 0.4Al under normal Al concentration in leaves, and there was a significant difference among different treatments (Fig. 9a). However, the application of high concentration Al showed no significant difference under early application of H2S or PAG (Fig. 9a). What is different in root is that except for the insignificant difference in LCD activity between H2S + 2Al, 2Al and PAG + 2Al, all other groups showed significant differences, and LCD activity showed H2S + Al > Al > PAG + Al (Fig. 9b).

      Figure 9. 

      C. sinensis on LCD activities in (a) leaves and (b) roots with different treatments. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

    • The synthesis of tea polyphenols was drastically promoted by H2S + 0.4Al, but slightly deduced by PAG + 0.4Al (Fig. 10a). Compared to 2Al, H2S + 2Al increased tea polyphenol content, while PAG + 2Al decreased tea polyphenol content, both of which were not significant (Fig. 10a). Similarly, the effects of various treatments based on 4Al on tea polyphenols were not significant (Fig. 10a).

      Figure 10. 

      The performance of (a) tea polyphenol, (b) free amino acid and (c) caffeine content in different treatments. Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.

      H2S + 0.4Al treatment induced the highest content of amino acids after treatment, significantly higher than both Al and PAG + 0.4Al (Fig. 10b). H2S + 2Al and H2S + 4Al did not significantly affect the amino acid content when compared to 2Al and 4Al, respectively (Fig. 10b). With PAG + 0.4Al treatment, amino acid content increased compared to 0.4Al, but amino acid content inhibited in PAG + 4Al, and no significant difference between PAG + 2Al and 2Al (Fig. 10b).

      Caffeine content at normal Al concentration showed no significant difference in caffeine content among H2S + 0.4Al, 0.4Al, PAG + 0.4Al. And H2S + 2Al, 2Al, PAG + 2Al were the same (Fig. 10c). The caffeine content only after being subjected to PAG + 4Al was greater than that of H2S + 4Al and 4Al (Fig. 10c).

      Results showed that the most abundant one was epicatechin (EC), along with of epigallocatechin (EGC), epigallocatechin gallate (EGCG), gallocatechin (GC), gallocatechin gallate (GCG), epicatechin gallate (ECG) and catechin (C) detected in tea leaves (Table 4). Compared to 0.4Al, H2S + 0.4Al increased the total catechin content by 9.48%, while H2S + 4Al has a 14.45% increase in total catechin content compared to 4Al (Table 4). In each component, GC and EGC under H2S + 2Al were increased compared to 2Al. C and EC contents can be generally stimulated under H2S + 0.4Al and PAG + 0.4Al, while C and EC contents were reduced by H2S + 4Al (Table 4). Although the contents of EGCG, ECG and GCG of ester catechins were relatively low, early application of H2S was still sufficient to stimulate an increase in EGCG, ECG, and GCG at 0.4Al and 2Al (Table 4). It was found that H2S + 0.4Al increased EGCG by 19.35% compared to 0.4Al, and H2S + 2Al increased EGCG by 8.70% compared to 2Al. Interestingly, even with early application of H2S, EGCG, ECG, and GCG were still repressed by 4Al (Table 4).

      Table 4.  Effect of different treatments on catechins in C. sinensis.

      TreatmentGC
      (%)
      EGC
      (%)
      C
      (%)
      EC
      (%)
      EGCG
      (%)
      ECG
      (%)
      GCG
      (%)
      Total catechins (%)
      0.4Al0.81 ± 0.03Aa2.92 ± 0.41Aa0.24 ± 0.00Ab3.68 ± 0.42Ab0.93 ± 0.09Ab0.38 ± 0.01Aa0.62 ± 0.06Ab9.81 ± 1.00Aa
      0.4Al + H2S0.80 ± 0.01Aa2.91 ± 0.06Aa0.248 ± 0.00Aa4.30 ± 0.17Aa1.11 ± 0.04Aa0.39 ± 0.00Aa0.73 ± 0.01Aa10.74 ± 0.16Aa
      0.4Al + PAG0.82 ± 0.01Aa2.44 ± 0.22Aa0.25 ± 0.00Aa3.95 ± 0.13Aab0.94 ± 0.05Ab0.37 ± 0.01Ab0.59 ± 0.04Ab9.61 ± 0.23Aa
      2Al0.76 ± 0.01Ba2.30 ± 0.35Aa0.24 ± 0.00Aa3.44 ± 0.29Aa0.92 ± 0.05Aa0.37 ± 0.01Aa0.60 ± 0.04Aa9.41 ± 1.30Aa
      2Al + H2S0.79 ± 0.02Aa2.80 ± 0.40Aa0.24 ± 0.01Ba3.35 ± 0.64Ba1.00 ± 0.15ABa0.37 ± 0.01Ba0.62 ± 0.07Ba8.91 ± 0.76ABa
      2Al + PAG0.76 ± 0.04Ba2.58 ± 0.31Aa0.24 ± 0.00Ba3.76 ± 0.62Aa1.06 ± 0.17Aa0.38 ± 0.01Aa0.67 ± 0.09Aa9.91 ± 1.19Aa
      4Al0.75 ± 0.02Ba2.31 ± 0.45Aab0.24 ± 0.01Aa3.59 ± 0.81Aab0.96 ± 0.11Ab0.37 ± 0.01Aa0.57 ± 0.06Ab7.82 ± 0.03Aab
      4Al + H2S0.77 ± 0.02Aa2.11 ± 0.09Bb0.23 ± 0.00Bb2.75 ± 0.04Bb0.82 ± 0.03Bb0.36 ± 0.00Ca0.53 ± 0.01Bb8.95 ± 1.57Bb
      4Al + PAG0.76 ± 0.01Ba2.80 ± 0.32Aa0.25 ± 0.00Aa4.11 ± 0.51Aa1.16 ± 0.11Aa0.33 ± 0.00Bb0.70 ± 0.04Aa10.26 ± 0.99Aa
      Data are mean values ± SD (n = 3). Different lowercase letters represent significant differences among different H2S conditions under the same Al concentration treatment, and different uppercase letters represent significant differences among different Al concentration treatments under the same H2S condition (p < 0.05), as determined by the Duncan test.
    • It is easy to accumulate too much availability Al3+ in the rhizosphere environment of C. sinensis suitable for planting in acid soil. Al actually has been regarded as an essential element with dose‐dependent effect, which is first reflected in root growth and development[3]. Root growth is stimulated in low concentrations of Al, while in high concentrations of Al, growth of the root and the plant is delayed[21]. In the present study, we also demonstrated that the effects on root development was strongly dependent on the Al concentration, the root system was damaged and new roots failed to generate by Al stress concentration (Fig. 1ai). At the same time, it showed that H2S broke the restriction of Al stress on root development, but PAG promoted the root development hindered by Al stress (Fig. 1ai). Moreover, pre-treatment with H2S increased total FW, total DW and root activity of C. sinensis to cope with excessive Al inhibition (Fig. 1j & 2). Recent research has demonstrated that H2S alleviates the inhibition of plant growth under metal stress in various crop plant species, including mungbean[22], soybean[23] and Miscanthus sacchariflorus[24]. These results indicated that H2S can effectively alleviate the growth and development of C. sinensis under Al stress.

    • Maintaining constant intracellular ion homeostasis is crucial for plants adapting to stress environments. Most of Al in C. sinensis was contained in root after Al stress (Table 2), affecting the root growth attributes more than the shoot growth attributes, which ultimately limited the growth and development of plants. Similar results were also observed in previous studies[25,26]. H2S alleviated the enrichment of Al in roots and promoted the TF of Al under Al stress, while PAG increased the accumulation of Al and inhibited the TF of Al (Table 2). Moreover, H2S application helped to maintain ion homeostasis by accumulating Ca in mature leaves, Mg, Zn in root and Mn in above-ground parts and increasing the TF of Fe under Al stress (Table 3). It has also been reported that H2S improves nutrients uptake under Al stress[27]. The results showed that H2S directly mitigated inhibitory effect of Al toxicity on root growth by decreasing content of Al in root systems, thus pre-application of H2S promoted the root growth and development of C. sinensis. Therefore, an increased uptake of Ca, Mg, Zn and Mn has been explained as a consequence of the stimulation of root growth under H2S.

    • We confirmed that excessive accumulation of Al disrupted ultrastructural and inhibited several processes, such as chlorophyll content and photosynthesis. Meanwhile, application of exogenous H2S enhanced chlorophyll content under Al stress conditions (Fig. 4), which was also reported by Ali et al.[27], who determined that H2S increased chlorophyll a and chlorophyll b by reducing damage to thylakoids in the chloroplast of Brassica napus. It is well known that the chlorophyll content and photosynthetic rate are closely correlated in plants. However, this result indicates that H2S failed to promote photosynthesis in C. sinensis under Al stress (Fig. 5), suggesting that H2S mitigates Al toxicity mainly through the increase of chlorophyll content and ultrastructural stabilization rather than regulating photosynthetic parameters.

    • Plants suffering from Al toxicity often exhibit symptoms associated with membrane lipid peroxidation, which result in accumulation of MDA[28]. As previously studied[29], the present results indicated that H2S reduce accumulation of MDA in leaves at 2Al (Fig. 6a). Proline participates in removal membrane lipid peroxidation under stress conditions[30]. However, using exogenous H2S at 2Al concentration only increases proline content in tea leaves by 2.82% compared to 2Al alone (Fig. 6b). CAT,POD and SOD are the main antioxidant enzymes in plants, all of which are involved in inhibition of oxidative stress and lipid peroxidation[31] in plants under excessive Al conditions, thus mitigating Al toxicity in plants[32]. CAT and POD played a role in the leaves under H2S + 2Al, because the activities of CAT and POD in H2S + 2Al were significantly higher than those in 2Al (Fig. 7a and 7c). There is also evidence indicating that H2S-induced alleviation in Al toxicity is attributed to elevated CAT and POD activities, but in barley roots[33]. At the same time, H2S + 2Al and H2S + 4Al reduced CAT, POD and SOD activities in roots, compared with 2Al and 4Al, respectively (Fig. 7b, d & f). When concerning reactive oxygen species scavenging systems, it is speculated that H2S may alleviate Al toxicity through elevated CAT and POD activities in leaves, while the root system mainly alleviates Al injury through other ways, thus the activities of CAT,POD and SOD decreased. Taken together this data supports the idea that H2S reduces MDA and increases proline levels by regulating antioxidant enzyme activity to alleviate stress in 2Al treatment in leaves.

      GSH, the major non-enzymatic antioxidants in the ASA-GSH cycle contribute to plant antioxidant defense[34]. Consistent with previous research results[35], the GSH content in leaves significantly increased after exposure to Al stress. Although exogenous H2S reduced the GSH content in barley leaves[35], it did not decrease GSH content in tea leaves under H2S + 2Al, and only decreased the GSH content under H2S + 4Al (Fig. 8a), indicating that H2S responds to 4Al toxicity by altering GSH content in leaves, triggering the AsA-GSH cycle and improving antioxidant capacity. Consistently, levels of GSSG, which is reduced to GSH, enhanced in leaves during Al stress exposure, and H2S reduce the content of GSSG only in 4Al (Fig. 8b). The GSH/GSSG ratio is also an important indicator of intracellular redox homeostasis within cells. Exogenous H2S modulated the GSH/GSSG ratios by altering GSH and GSSG to varying levels, but resulting in a little change in GSH/GSSG compared to Al stress alone (Fig. 8c). These outcomes are consistent with the findings of previous studies on bermudagrass[36] and rice[37]. GST has been found to catalyze the chelation of GSH with metals and reduce the toxicity of metals to plants[38]. The GST activity under H2S + 2Al not H2S + 4Al stress was significantly enhanced (Fig. 8d), plants rely on the binding to minimize damage, which was consistent with the study of Miscanthus sacchariflorus[24].

      GR regulates the redox state of glutathione by converting GSSG into GSH, and also responsible for combating a large amount of reactive oxygen species in plants[39]. The GR activity in this study shown an increase under Al stress which is similar to the observations made by Devi et al.[40]. Higher GR activity after H2S + 2Al and lower GR activity under H2S + 4Al were observed, respectively in comparison to 2Al and 4Al (Fig. 8e). The above results confirmed that H2S alleviates 2Al stress by regulating substances derived from antioxidant system, whereas the mechanism was complex, resulting in a small pattern of changes in H2S + 4Al compared with 4Al stress alone.

      LCD is primarily responsible for catalyses the decomposition of cysteine to H2S. Further enzyme analysis indicated that the externally applied H2S enhanced the activity of LCD relative to Al alone stress, which was especially significant in roots. In Spinacia oleracea also clearly showed an increase in LCD activity with application H2S[41], and an early H2S signal might promoted higher LCD activity than Al stress after 3 h[42]. Taken together, LCD activity regulates the internal H2S pathway in C. sinensis and plays a more effective role in roots rather than leaves

    • Various components of the tea plant, including tea polyphenols, amino acids, caffeine, catechins, are not only closely related to the flavor of the tea plant, but also have an effect when C. sinensis is exposed to stress. The synthesis of amino acids, caffeine, and catechins is regulated by Al[43]. In this study, compared with normal Al concentration, the changes in tea polyphenol content under Al stress were not significant, while the content of free amino acids was significantly reduced and the content of caffeine was significantly increased (Fig. 9). At normal Al concentration, early application of H2S increases the content of these substances (Fig. 9), which may be related to the promotion of tea roots growth by H2S[44]. As a major component of the ester type catechins, EGCG has been reported to chelate Al, thus conferring Al tolerance to plants[45]. It was found that the EGCG content increased by 8.70% under H2S + 2Al compared to 2Al, and excessive stress of 4Al may lead to a decrease in EGCG content, and even with the addition of exogenous H2S, the changes in content remains small under 4Al stress (Table 4). Combined with the above results, it providing further evidence that the part of H2S that promotes the increase of components may have chelated with too much Al at H2S + Al, resulting in a decrease in the final content, or may be caused by severe stress at 4Al.

    • Our results indicate that H2S may be pivotal actor in enhancing the resistance of C. sinensis to Al stress. Increasing biomass, promoting root activity, reducing accumulation of Al in roots and increasing TF of Al, regulating the content of Ca, Mg, Zn, Mn and Fe and their TF in different tissues, increasing chlorophyll content, maintaining ultrastructural homeostasis, regulating substances related to antioxidant pathways and tea plant components all play key roles in the ameliorating effect. Moreover, compared to 4Al, H2S can better alleviate the stress caused by 2Al.

    • The authors confirm contribution to the paper as follows: conceptualization: Shu Z, Sui X, Wang Y; investigation: Shu Z, Huang P, Wan S; data curation: Zhang Y, Xing A; project administration: Shu Z, Wang Y; supervision: Wang Y; resources: Li X; formal analysis, visualization, writing—original draft preparation: Xing A; writing—review and editing: Xing A, Liu S, Chen X, Li X, Wang Y; funding acquisition: Chen X, Wang Y. All authors read and approved the final manuscript.

    • All data generated or analyzed during this study are available within the article.

      • We thank Professor Yanjie Xie from College of Life Sciences, Nanjing Agricultural University, for revising the paper. We thank Dr. Yuehua Ma (Central Laboratory of College of Horticulture, Nanjing Agricultural University) for using Microporous Plate Detecting Instrument (Cytation3, BioteK, USA). This work was supported by the National Natural Science Foundation of China (31972457), Jiangsu Agricultural Industry Technology System (JATS[2023]416), and Natural Resources Science and Technology Foundation of Jiangsu Province (2022018).

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Anqi Xing, Zaifa Shu

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (10)  Table (4) References (45)
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    Xing A, Shu Z, Huang P, Zhang Y, Sui X, et al. 2024. Exogenous hydrogen sulfide enhanced Al stress tolerance in tea plant Camellia sinensis. Beverage Plant Research 4: e024 doi: 10.48130/bpr-0024-0013
    Xing A, Shu Z, Huang P, Zhang Y, Sui X, et al. 2024. Exogenous hydrogen sulfide enhanced Al stress tolerance in tea plant Camellia sinensis. Beverage Plant Research 4: e024 doi: 10.48130/bpr-0024-0013

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